By Leah Davis
“The meeting was excellent. I am so pleased we organised it to fit in with Prof Resia Pretorius’ visit to Oxford. We rarely get together as group of collaborators working on fatigue. The meeting was warm and open, and I hope exciting collaborations can come from it. Our social dinner in the evening was even “warmer” as we moved even deeper into the period of hot weather. All the speakers have put together Podcasts about their work to support our IBRO project which we will put on line shortly”Karl Morten
Link between fatigue and blood brain barrier breakdown in rodent models – Associate Professor Brad Sutherland, University of Tasmania
Associate Professor Brad Sutherland reported on the importance of the neurovascular unit in the maintenance of brain health through its contribution to the blood brain barrier (BBB), increasing intracellular communication and controlling blood flow in response to stimulation.
Disruption to this neurovascular unit can result in disease; in those with ME/CFS chronic inflammation is thought to lead to a breakdown of the neurovascular unit that can increase permeability of the blood vessels and restrict cerebral blood flow. Brad discussed research whereby rodent models with induced inflammation either by lipopolysaccharide (LPS) or poly I:C infusion are shown to have abnormal neurovascular cells, reduced Glut1 levels within endothelial cells, and damaged astrocyte end-feet. Reduction in Glut1 levels results in a reduction in glucose levels entering the brain, which could contribute to the fatigue present.
Damage to the BBB was evident by staining for fibrinogen, which was shown to leak into the brain due to vessel changes induced by LPS. Lastly Brad showed that transgenic mice models with depleted pericyte numbers resulted in reduced movement within an open field study, indicating fatigue-like symptoms. Brad concluded that pericytes are vital to brain health, with loss of these cells resulting in damage to the neurovascular unit, resulting in fatigue-like symptoms possibly due to the loss of vessels that provide the brain with the energy required for normal function.
Microclots in Long Covid and links to pathology – Professor Resia Pretorius, University of Stellenbosh
Professor Resia Pretorius reported on the links between the formation of microclots and the development of Long Covid. Inflammatory molecules that affect blood clotting, including platelets, red blood cells and fibrinogen, are thought to play a role in the pathology of Long Covid, with abnormalities within the clotting process leading to the formation of microclots within the blood.
Structural changes occur within fibrinogen due to oxidative stress factors, present as a result of circulating inflammatory molecules and viral spike protein which bind the fibrinogen and alter its structure, potentially converting the alpha helix structures into beta sheets; it is this change in structure that leads to the abnormal clotting observed in those with the virus. Abnormal clotting has been observed in those with acute Sars-CoV-2 infection, with all ICU patients being shown to have damaged cellular membranes, resulting in the secretion of cellular contents which complex with the red blood cells affecting blood viscosity.
The Sars-CoV2 spike protein has been identified as amyloidogenic, interacting with fibrinogen to form clot like structures. Resia reported that these microclots have been found at high levels within those suffering acute Sars-CoV-2 infection and suggested it was the failure to clear these clots that contributes to the development of Long Covid. In 10-30% of those who suffer acute infection, the fibrinogen system is thought to fail, resulting in an inability to clear the microclot.
When the clot contents was analysed, compounds such as alpha-2-antiplasmin were found rendered inside, preventing it from conducting its role in clot breakdown. Resia thus concluded that persistence of these microclots contribute to the pathology of Long Covid.
Chronic Ischaemia-Reperfusion injury in Long Covid resulting from fibrin amyloid microclots – Professor Douglas Kell, University of Liverpool:
Professor Douglas Kell reported on the risks of chronic Ischaemia Reperfusion injury in Long Covid. Symptoms of Long Covid have been shown to include fatigue, malaise, decreased blood O2 and blood clots. These blood clots have been identified to contain an amyloid component generated by the S1 spike protein, which induces a fibrin-amyloid microclot formation.
Clots that form are more resistant to the fibrinolytic system, so the body struggles to break them down. As a consequence these microclots end up stuck within the capillary networks, preventing red blood cells reaching the fine tissue capillaries, resulting in ischemia due to lack of oxygen exchange. The tissue becomes ischaemic at the site of the block and once blood returns and oxygen is delivered there is the possibility of localised reperfusion injury as the mitochondrial respiratory chain returns to normal function. However, being in an over-reduced state it now forms bursts of cytotoxic reactive oxygen species (ROSs).
Similar clots have been discovered in ME/CFS, but are less extensive. Doug suggested that during this ischemic episode, reactions such as the Fenton and Haber Weiss occur, resulting in the production of superoxides and hydroxyl radicals; once reperfusion occurs these are released, killing cells and impairing cellular function.
Doug stated reperfusion may help to explain the unusual kinetics seen within those suffering post-exertional malaise, with there being a time delay between the production of the ROS and the cellular damage as a consequence of reperfusion. Doug summarised that it is possible the microclots are inducing ischemic periods in people suffering with Long Covid by preventing blood flow, generating harmful species that lead to reperfusion injuries when blood flow resumes. It is also thought that Omicron is less amyloidogenic, resulting in fewer of these clots forming, which may explain its reduction in the severity of symptoms.
Developing new diagnostic tests for chronic diseases – Professor Karl Morten, University of Oxford
Professor Karl Morten reported on the importance of developing a diagnostic test for chronic conditions, to ensure the best outcome for patients with ME/CFS. Attempts have been made to utilise plasma metabolomics as a method to identify potential biomarkers. Metabolomics provides a profile of the chemical components present within the plasma of an individual, allowing for comparisons between healthy individuals and patients. This technique was used to study two cohorts, a Polish cohort, including 54 mild/moderate ME/CFS patients and 23 healthy controls, and a larger ME UK Biobank study, involving 150 Mild, Moderate and Severe ME/CFS patients, 50 healthy controls, and 50 Multiple Sclerosis (MS) patients for comparison.
Within the Polish cohort, metabolomics modelling showed promise, with PLSDA plots demonstrating good separation of patients and controls. 1168 metabolites were shown to be significantly different between ME/CFS patients and controls with a clear separation in the metabolomics profiles, however most of these chemicals, including the top 25 most significant, are currently unknown. With the UK ME Biobank data however, the ME/CFS V control modelling was far weaker, with no separation observed. This may be due to several factors, including processing times and diagnosis. Despite this the data did indicate a potential for liver issues within the ME/CFS patients, with dysregulation occurring within the urea cycle, as well as with taurine metabolism and the kynurenine pathway, providing potential avenues that can be explored further. There are issues associated with this technique however, with confounding factors including age, sex and BMI having a potential influence on the metabolome.
Other components in the blood, such as Peripheral blood mononuclear cells (PBMCs) and plasma extracellular vesicles, can be studied using Raman Spectroscopy, a technique that provides information about the chemical structures of samples through profiling molecular bond vibration. Raman micro-spectroscopy has the potential to be developed as a diagnostic tool, and was utilised to study the PBMCs of 100 subjects with ME/CFS and MS, as well as healthy controls.
Using this technique on samples from severe ME/CFS and healthy controls, extracellular vesicles (EV’s) and MiRNA from PBMCs and Raman spectral profiles of EV’s were identified as key parameters which could separate the two groups. Within these EV, carotenoid peaks were identified, which are found in red blood cell ghosts, suggesting major EV population may be RBC derived. RBC ghosts are seen in later studies in chronic disease patients in combination with inclusions which were found to increase over time when fresh blood was left on a slide.
Identifying factors in the blood which impact cellular energetics in ME/CFS and developing a diagnostic assay – Miss Edie Guo, University of Oxford
Chronic Fatigue Syndrome, a chronic condition characterised by several symptoms including debilitating fatigue that does not improve with rest, is thought to affect 20 million individuals across the globe. Despite its prevalence however, the condition still lacks a specific biomarker, so no diagnostic test has been discovered, which consequently limits diagnoses of the condition.
CFS has a similar symptom profile to other chronic conditions, including Chronic Lyme (muscle aches and severe headaches) and Long Covid (joint pain, fatigue, malaise and depression/anxiety). As a result of the lack of diagnostic test, this project aims to identify factors within the blood that impact cellular energetics in ME/CFS, such as the contents of plasma, viruses and other molecules, using these to develop a diagnostic assay for the condition through platforms such as Raman spectroscopy and Intelligent Lab on Fiber systems. Different patient cohorts are being utilised, including patients who have undergone whole-body cryo-stimulation, which has been shown to decrease fatigue and aortic stiffness until 3 months (reverts back after 12 months), and Lyme samples from patients who have been given a long course (2 month) duration of antibiotic treatment. The impact of the plasma factor will be studies in two different cell types were also studied: Human Umbilical Vein Endothelial Cells (HUVECs) and Primary muscle cells.
Within ME/CFS, plasma from these patients has been shown to reduce the ability of these endothelial cells to produce nitric oxide; decreased NO production has been linked to an increase in inhibitory phosphorylation of ENOS at Thr495 basal site. These cells are also the first place of contact for blood cells and plasma. Muscle dysfunction in ME/CFS, as well as inflammation and activation of cell mediated immunity indicate primary muscle cells may also be beneficial to study further.
A protocol to investigate brain, mind and body contributions to causing Fatigue in ME/CFS and Long COVID – Dr Inga Williams, University of Oxford
Very few studies have a longitudinal design to test what may cause Fatigue. Inga presented her protocol to begin to remedy this by using longitudinal approach to investigate possible causes of Fatigue in ME/CFS and Long COVID patients.
Studying ME/CFS using plasma metabolomics – Mr Jamie Strong, University of Oxford
Metabolomics can be used to determine changes in metabolite abundances which may underpin specific disease phenotypes, such as severity of specific neurosensory symptoms such as hyperacusis. Although this method does provide insight into what is happening inside the body, overall modelling (looking at all metabolites together (the metabolome), does not assess enzymatic flux of substrates, such as lactate relative to pyruvate (via Lactate Dehydrogenase) and glutamate to glutamine, which may be essential to providing in-depth clues as to which proteins may be disrupted in a disease.
With observational data obtained from patient intakes during routine clinical practice, demographic factors such as Body Mass Index (BMI), age, medication and sex are seldom matched and can therefore have profound influence on the plasma metabolic profile if models containing impacted metabolites are not sufficiently adjusted for using regression techniques.
Mass Spectrometer instrument drift can also impact on the abundance of metabolites, similarly to that of demographic factors. However, this impact can be overcome through adjustments of metabolite drift using published Excel based tool called MetaboDrift. Two cohorts of ME/CFS patients were studied, a Polish cohort and a ME UK Biobank cohort, using metabolomics to determine the metabolite profile of plasma, with the former cohort being compared to healthy controls and the latter compared to both healthy controls and Multiple Sclerosis subjects with co-morbid fatigue. Within the Polish cohort, the amino acids showed the greatest difference between ME/CFS patients and healthy controls when using Partial Least Squares Discriminant Analysis (PLS-DA) analysis.
Upon further study, all of the top 25 significant metabolites contributing to the predictive capability of the model were of unknown identity. These ME versus control differences (in the Polish Cohort) were strengthened when we compared a group of ME/CFS patients who having completed a structured exercise programme over the course of around 6 months, demonstrated relative to the control group at baseline – a greater separation between healthy controls and ME/CFS patients. Some identified metabolites in the Polish cohort included alterations in the abundance of glutamine, which was down around 50% in patients, as well as a 50% reduction in cysteine, a substrate for glutathione synthesis. Other amino acids affected include methionine, glycine, serine and alanine, which are all glucogenic TCA substrates, and threonine, a glucogenic and ketogenic substrate. To combat the issues associated with metabolomics and strengthen the data produced from MetaboAnalyst, the development of an Excel based tool, MetaboOutlier, has been explored to identify metabolites on a per patient basis that are far from the norm of healthy controls, measuring for example standard deviations relative to a mean value. This software then generates a per patient metabolic severity score, which can be compared to clinical findings to determine its relevance (enabling assessment beyond correlations with only single metabolites).
The miracle of Microbiome mapping – Dr Julian Kenyon, The DoveClinic
Dr Julian Kenyon reported on the importance of the microbiome within both chronic conditions and cancer, with alterations to the community of gut microorganisms being shown to play a role in these disorders. Discussed was a retrospective study involving 42 patients with CFS, 30 of which also had IBS. The cohort was split into two, with half treated orally (probiotics, nutritional remedies etc), while the others were treated with Faecal Microbiome Transplantation via paediatric rectal catheter.
Results showed that the FMT group showed a much greater improvement, with the median being found to be much higher in this group compared to the oral treatment group, demonstrating FMT is a safe and promising treatment for CFS associated with IBS, and highlighting an area where randomised controlled trials should be carried out. Julian also reported on the importance of the microbiome within the oncology field; patients were recruited to the study, with stool samples being sent to Atlas Biomed for 16S RNA sequencing to determine values for microbiome diversity, probiotic bacteria, and fibre and butyrate producing bacterial species. Values for these categories were shown to be different across several conditions including cancer, CFS and IBS, demonstrating the potential importance of a dysbiotic gut in these diseases.
It was also shown that the poorer the quality of the microbiome mapping, the worse the prognosis appeared to be for the cancer patient; improving the microbiome mapping could potentially improve prognosis. It is well known that the gut microbiome also impacts on developing of malignancies, as well as the success of cancer therapies, including immunotherapies. Treatments to re-stabilise the microbiome, such as Gut Flora Replacement Therapy, should be explored further for cancer patients. GFRT has been used in the context of ME/CFS, with 10 randomly chosen FMT patients and 10 randomly chosen patients who undertook GFRT being compared using 16s MRNA microbiome mapping; GFRT was reported to be just as effective, but also safer than FMT.
Effects of whole-body cryotherapy and static stretching are maintained after 4 weeks of treatment in patients with chronic fatigue syndrome – Dr Slawomir Kujawski, Nicolaus Copernicus University
A symptom specific approach to understanding CFS and determining potential treatment strategies may be a valuable method to improve understanding of the condition. Brain fog, shown to affect 50-85% of CFS suffers, as well as autonomic nervous system disturbances and chronic inflammation, are all correlated with symptoms found in patients with the condition. It is thought that Whole-Body Cryotherapy (WBC) may be an effective treatment for these symptoms. Studies conducted by Ginzinska et al (2015), demonstrated WBC could decrease inflammatory molecules such as IL-6 and TNF-alpha in those suffering from Rheumatoid Arthritis, while Rymaszewska et al (2018) showed WBC has a beneficial effect on cognitive function, increasing performance in memory tests.
Based on this knowledge, a study was conducted, with 32 CFS patients being enrolled in the study after meeting the inclusion criteria. All of these patients were followed up just after WBC, however only 22 were analysed 4 weeks after WBC as 10 were lost to follow-up. Data was collected pre-WBC, to gain insight on autonomic nervous system function, as well as cognitive function, for comparison after the programme. The WBC programme consisted of 10 sessions, with each chamber exposure increasing across these sessions from 30-150 seconds. This study indicated that WBC could reduce fatigue and symptom severity related to dysfunction within the autonomic NS, as well as improve cognitive function 4 weeks after the programme ended, with it being thought WBC may be beneficial in decreasing the severity of these symptoms in low to moderate ME/CFS patients.
It is worth noting however that 10 patients did not attend the follow up, with these being those in the higher fatigue groups, thus the long term effects of the WBC should be interpreted carefully. Therefore, the current pilot study needs to be replicated before drawing conclusions in terms of application to patients.
Brain and muscle imaging in ME/CFS and Long Covid – Dr Beata Godlewska and Professor Ladslav Valkovič, University of Oxford
Long Covid and ME/CFS both have a plethora of symptoms, with some being shared between the two conditions, such as fatigue and cognitive dysfunction (‘brain fog’). A shared ‘initial injury’ for both conditions is thought to be a viral infection, with subsequent systemic inflammation and sickness. In most cases, this returns to normal, however in a small proportion of people this immune response stimulates processes that lead to the development of long-term and often debilitating fatigue and related symptoms.
Magnetic Resonance Spectroscopy (MRS) can be used as an in vivo tool to analyse the biological changes that underpin both conditions, allowing researching diagnostic and treatment biomarkers, as well as for the similarities and differences between both conditions. We are currently conducting two studies using MRS The first uses 7T MRS to determine both the brain and muscle biochemistry of CFS and Long Covid and how this may link to symptoms expressed, while the second, using 3T MRS, assesses creatine supplementation as a treatment for CFS. The data is still being collected for both studies, with the aim of 30 participants in each group for the 7T MRS study (until now, 19 patients with CFS and 20 patients with Long Covid were included) and 15 participants in the creatine supplementation study.
The preliminary results for the 7T MRS study suggested increased levels of choline containing compounds, phosphocholine and glycerophosphocholine. Choline compounds are considered a marker of inflammation in the brain (their production increases with increased gliosis and related rapid membrane turnover). In our study, those with Long Covid, we saw a relationship between choline compounds level and performance on the Tower of London, a task assessing executive function.. Increased inflammation can lead to a cycle of glutamate toxicity, oxidative stress and mitochondrial damage, resulting in dysfunctions in energy production in the cells across the body.One of the markers of abnormal energy production are elevated levels of lactate. In our 7T MRS study, we observed increased levels of lactate in the brain of both CFS and Long Covid patients. We have also seen increased levels of glutamate in patients in CFS.
The above results are interesting in the context of the above mentioned potential mechanisms of both conditions. However, at this point, the results need to be treated as preliminary until the study is completed. Another marker of abnormal energy metabolism is decreased levels of creatine, seen in some of the previous studies, suggesting further studies into creatine supplementation may be beneficial. The second study we are currently running, aims at an assessment of the impact of oral creatine supplementation (16g daily for 6 weeks) on brain creatine levels, , muscle strength, cognition and activity (ActiWatch) in volunteers with CFS.. Data from 12 participants has been collected up to date and analysis is ongoing.
From Ignorance to Activism: the perils of criticising establishment-backed treatment studies in ME/CFS – Ms Caroline Struthers, University of Oxford:
Caroline reported on her experiences working for and interacting with medical research institutes such as Cochrane. Scientific studies, including systematic reviews, designed to provide evidence for the effectiveness of healthcare treatments are often given more credibility if they report results supporting the establishment view, or the interests of the researchers carrying out the studies. She has discovered how study methods can be manipulated to flatter favoured interventions and how researcher allegiance bias, particularly of psychological and behavioural interventions, is routinely ignored.
Using ME/CFS and exercise therapy as an example, Caroline pointed out that the needs of those with vested interests in generating scientific evidence to support their long-held theories and beliefs has been prioritized over the need to protect patients from ineffective and potentially harmful treatments.